Our laboratory has developed new methods for disease discovery and identified the molecular bases for three fatal neurodegenerative disorders. This work grew out of our basic research on lysosomal enzyme targeting.
Lysosomes are membrane-bound, acidic organelles that are found in all eukaryotic cells. They contain a variety of different proteases, glycosidases, lipases, phosphatases, nucleases and other hydrolytic enzymes, most of which are delivered to the lysosome by the mannose 6-phosphate targeting system. In this pathway, lysosomal enzymes are recognized as different from other glycoproteins and are selectively phosphorylated on mannose residues. The mannose 6-phosphate serves as a recognition marker that allows the enzymes to bind mannose 6-phosphate receptor which ferry the lysosomal enzyme to the lysosome. In the lysosome, the enzymes function in concert to break down complex biological macromolecules into simple components. The importance of these enzymes is underscored by the identification of over thirty lysosomal storage disorders (e.g., Tay Sach's disease) where loss of a single lysosomal enzyme leads to severe health problems including neurodegeneration, progressive mental retardation, and early death. There are also a number of unsolved genetic diseases that are likely to arise from deficiencies in as yet undiscovered lysosomal enzymes.
Our approach to identify the molecular basis for unsolved lysosomal storage disorders is based on our ability to use mannose 6-phosphate receptor derivatives to visualize and purify mannose 6-phosphate containing lysosomal enzymes. For instance, we can selectively compare the spectrum of lysosomal enzymes present in normal and disease specimens. If the disease specimen lacks a given lysosomal protein, this may be responsible for disease. To investigate this, we purify and sequence the normal protein, clone the corresponding gene, and examine patients for mutations associated with disease. In this manner, we found that a fatal childhood neurodegenerative disease called LINCL (late infantile neuronal ceroid lipofuscinosis) is caused by mutations in a gene encoding a previously undiscovered lysosomal protease, now known to be tripeptidyl-peptidase I (TPP I).
LINCL is literally a disease from hell, as parents see what has been a normally developing child degenerate before their eyes. Children typically develop normally until age 3 at which point they exhibit ataxia and seizures. They start losing vision a year later, and within a few years are blind, mute, and completely bedridden. The children usually die between ages eight and fifteen, although there are some mutations that result in a later-onset, prolonged disease. At the cellular level there is extensive lysosomal accumulation of autofluorescent storage material (ceroid lipofuscin) accompanied by massive death of neurons and marked brain atrophy. About fifty children are diagnosed with LINCL each year in the United States and the disease has devastating effects on the affected children and families.
While our laboratory primarily conducts basic research, our interactions with many LINCL families have given us added impetus to extend our research to the clinic. After we identified the gene and determined the function of corresponding protein, we developed rapid biochemical and DNA-based assays for definitive pre-and postnatal diagnosis and carrier screening. This allows for genetic counseling to prevent further occurrence of the disease. However, in the absence of universal carrier testing, new cases will continue to arise so it is important to develop effective therapies that can halt and reverse disease progression. To this end, we have produced recombinant enzyme in a form that can be taken up by affected cells in culture to correct the primary defect. We have also developed a LINCL mouse model and are using this to investigate the biological function of the TPP I protein as well as to evaluate potential therapeutics strategies.
Another research program in the laboratory is to identify the spectrum of lysosomal enzymes encoded by the human genome. This research is particularly timely given the current effort towards determining the complete sequence of the human genome. Our approach is to purify mannose 6-phosphorylated proteins by affinity chromatography, determine their identity using different proteomic approaches, and verify their subcellular localization. We have currently identified a number of new lysosomal proteins and are working to characterize their role in biology and medicine. We recently used this approach to determine the molecular basis for Niemann Pick type C2 disease, a fatal cholesterol storage disorder.
- Sleat DE, Donnelly RJ, Lackland H, Liu CG, Sohar I, Pullarkat RK, Lobel P.(1997)Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis. Science 277:1802-5
- Tyynela J, Sohar I, Sleat DE, Gin RM, Donnelly RJ, Baumann M, Haltia M, Lobel P. (2000)A mutation in the ovine cathepsin D gene causes a congenital lysosomal storage disease with profound neurodegeneration. EMBO J 19:2786-92
- Naureckiene S, Sleat DE, Lackland H, Fensom A, Vanier MT, Wattiaux R, Jadot M, Lobel P. (2000)Identification of HE1 as the second gene of Niemann-Pick C disease. Science 290:2298-301
- Lin L, Sohar I, Lackland H, Lobel P. (2001)The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH. J Biol Chem 276:2249-55
- Sleat DE, Wiseman JA, El-Banna M, Price SM, Verot L, Shen MM, Tint GS, Vanier MT, Walkley SU, Lobel P. (2004) Genetic evidence for nonredundant functional cooperativity between NPC1 and NPC2 in lipid transport. Proc Natl Acad Sci U S A. 101:5886-91 PMID: 15071184
- Sleat DE, Wiseman JA, El-Banna M, Kim KH, Mao Q, Price S, Macauley SL, Sidman RL, Shen MM, Zhao Q, Passini MA, Davidson BL, Stewart GR, Lobel P. (2004) A mouse model of classical late-infantile neuronal ceroid lipofuscinosis based on targeted disruption of the CLN2 gene results in a loss of tripeptidyl-peptidase I activity and progressive neurodegeneration. J Neurosci.24:9117-26 PMID: 15483130
- Sleat DE, Lackland H, Wang Y, Sohar I, Xiao G, Li H, Lobel P. (2005) The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins. Proteomics. 2005 5:1520-32. PMID: 15789345